A Novel Panel of 36 Insertion/Deletion (InDel) Markers for Human Identification

doi:10.21203/rs.3.rs-1850086/v1

Background: In recent years, the insertion/deletion (InDel) polymorphism has become a preferred genetic marker in forensic genetics due to its low mutation rates and small amplicon sizes. In this study, a 36-InDelplex identification panel, consisting of autosomal 34 InDel loci, 1 Y InDel locus, and amelogenin, was developed and gene frequencies in the Turkish population were determined.

Methods and Results: The loci of the InDel panel with global minimum allele frequencies (MAF) ≥ 0.4 were selected from the 1000 Genomes Project Phase 3 data. The amplicon sizes of the loci were designed to be smaller than 250 bp. In the validation study of the created panel, analysis threshold, dynamic range, sensitivity, stochastic threshold, inhibitor tolerance, and reproducibility parameters were studied in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) guideline. The sensitivity studies indicated that complete and reliable InDel profiles could be obtained with 0.25 ng of DNA. The 36-InDelplex panel was evaluated using the 250 samples from individuals of Turkey. The mean observed heterozygosity ratio (Ho) of all loci was 0.48. Combined discrimination power (CPD) is 0.999999999999988 and combined exclusion probability (CPE) is 0.9987. The population comparison also made using Turkish and each of the five major populations 1000 Genomes Phase 3 populations’ data for each of the five major populations (i.e., African, European, East Asian, South Asian, and American).

Conclusions: The results showed that the 36-InDelplex panel is a reliable, sensitive and accurate system, which is suitable for human identification and population genetics purposes.

Insertion/Deletion (InDel) polymorphism

Human Identification

Forensic Genetics

Panel Development

Degraded DNA Analysis

Population Genetics

STRs (Short Tandem Repeats) have been widely used in forensic genetics due to their high polymorphism and ease of analysis [1, 2]. However, successful profiling is often not possible when there are small amounts and/or degraded biological samples from the scene. In recent years, different polymorphic systems have been sought to overcome such problems [3]. SNPs (Single Nucleotide Polymorphism) and InDels (insertions/deletions) are used in the identification of degraded samples by taking advantage of their short amplicon sizes. SNPs and InDels are also frequently applied in kinship analysis because of their low mutation rate [4]. Polymorphic systems with a low mutation rate are more stable in paternity and kinship tests, so the probability of false exclusion is low [5, 6]. SNP analysis requires complex and time-consuming additional procedures (e.g. SNaPshot technic) compared to InDels which can be employed standard techniques such as PCR with fluorochromelabelled primers and capillary electrophoresis. Thus, Indels combine the advantages of STR and SNP loci and can be used as an alternative or additional to these loci [7].

InDels are a type of polymorphism that occurs as insertions or deletions of nucleotide sequences in the genome [8, 9]. 1.4 million InDels have been characterized by the human genome project. Weber et al. showed that approximately 2000 of them are diallelic and are found every 7.2 kb in both the coding and non-coding regions of the human DNA [10, 11]. Since InDels have small amplicon lengths (< 260 bp), successful profiling is possible in small amounts and/or degraded DNA samples [12]. Although InDels have low discrimination power compared to STRs, extended panels with high discrimination power can be created with their capability of high-throughput genotyping systems [13]. Several InDel identification panels have been developed by various researchers [13–19].

In this study, we developed and validated a novel 36-InDelplex panel, consisting of 34 autosomal InDels and 1 Y-InDel, and Amelogenin as sex markers, for the use of forensic identification and kinship analysis. The PCR-based 36-InDelplex loci are shorter than 250 bp improve genotyping success in the low amount, and degraded for forensic samples.

2.1 DNA Samples

In this study, blood or buccal swab samples were collected along with signed informed consent from 250 unrelated, healthy, and randomly selected volunteers (125 males and 125 females) from the Turkish. All of the selected volunteers and their at least three generations families originated from Turkey. We also compiled reference population samples from the Ensemble (www.ensemble.org) database of 1000 Genome Phase 3 containing African (N = 661), European (N = 503), American (N = 347), South Asian (N = 489), and East Asian (N = 504) reference population data. K562 (10 ng/µL) (Promega) control DNA sample was used for optimization and validation of the 36-InDelplex panel.

DNAs were extracted using the Invitrogen™ PureLink-Genomic DNA Mini Kit (Thermo Fisher Scientific, MA, USA). DNA amounts were determined with the fluorometric Qubit™ dsDNA HS Assay Kit according to the manufacturer's recommendations (Thermo Fisher Scientific, MA, USA).

2.2 Locus Selection and Primer Design

An initial set of candidate InDel loci were selected from the available online database of the NCBI database (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/), the Ensemble database (https://www.ensembl.org/info/genome/variation)/species)/populations.html).

All candidate InDel markers were selected considering the following criteria: (i) InDel located in non-coding regions of the autosomal chromosomes; (ii) loci with minimum allele frequencies (MAF) ≥ 0.4 and heterozygous ratios ≥ 0.5 in all populations based on Phase 3 data of the 1000 Genomes Project; (iii) allele length variation at 2–10 bp range; (iv) InDel loci with a distance ≥ 200 Kb. Additionally, both Y InDel and Amelogenin markers were selected for gender in the 36-InDelplex panel. Detailed information on all selected markers is listed in Table 1.

Table 1

Selected InDel Loci and features for the 36-InDelplex Panel
No	rs Number	Alleles Genomic	Chromosome	Location	Fluorescent Dye
ID1	rs4646006	-/AATG/AGTG/CGTG	1	15518528:15518529	ATTO-550
ID2	rs2307838	-/TGAA	1	55749938:55749945	6-FAM
ID3	rs2307521	-/A/AG/CG	1	205259305:205259306	6-FAM
ID4	rs28369942	-/CAAGT	2	99465100:99465103	ATTO-550
ID5	rs140861207	-/AG	3	42996121:42996122	JOE
ID6	rs1160965	-/TG	5	14193899:14193903	ATTO-565
ID7	rs2307656	-/GTTAA/GTTAA	5	34844320:34844327	6-FAM
ID8	rs2067304	-/GA	5	61176982:61176987	6-FAM
ID9	rs1610937	-/AAGG	5	77449242:77449252	6-FAM
ID10	rs10590424	-/AATAA	5	80051331:80051340	JOE
ID11	rs1160981	-/CAG	5	131329321:131329326	ATTO-565
ID12	rs2067191	-/ATAG	5	135938900:135938902	ATTO-565
ID13	rs144389514	-/ATCT	6	8507865:8507868	ATTO-550
ID14	rs2308137	-/GA	6	149293062:149293067	JOE
ID15	rs16646	-/CTTT	7	103760898:103760900	6-FAM
ID16	rs16458	-/CTTC	7	122511274:122511280	ATTO-550
ID17	rs3062629	-/TACAG	8	10748710:10748714	ATTO-565
ID18	rs2308072	-/AGGA	8	19232270:19232274	ATTO-550
ID19	rs10623496	-/GAAT	8	122933405:122933406	ATTO-565
ID20	rs56168866	-/A/AA/CA/CCA	9	9246999:9247000	ATTO-565
ID21	rs2308112	-/TGTGG	9	95811827:95811833	ATTO-550
ID22	rs2308135	-/TAA	11	66628515:66628517	ATTO-550
ID23	rs33972805	-/CT/T	11	126418977:126418978	JOE
ID24	rs1610919	-/TA	12	14757062:14757064	ATTO-565
ID25	rs34660708	-/TG	13	23391809:23391812	6-FAM
ID26	rs2308163	-/ATTG	14	57583364:57583369	JOE
ID27	rs2067147	-/ATC	14	71867438:71867444	ATTO-550
ID28	rs16722	-/TGTT	17	11448230	ATTO-565
ID29	rs25549	-/CATT	17	59951249:59951256	ATTO-550
ID30	rs34495360	-/GACTT	20	4973464:4973468	JOE
ID31	rs16671	-/GG/GGG	20	9518908:9518910	6-FAM
ID32	rs2308101	-/TA	20	52629056:52629057	JOE
ID33	rs6480	-/GT	22	35314218:35314220	JOE
ID34	rs16363	-/TTGTT/(TTGTT)2	22	37013845:37013853	JOE
ID35	rs2032678	-/TTCTC	Y	13396820:13396830	6-FAM
ID36	AMG-XY	X/Y	XX-XY		6-FAM

The primer design of the selected InDel loci was such that (i) average primer sequence lengths were between 20–26 bp, (ii) amplicon lengths were in the range of 70–250 bp, (iii) GC% ratios in the primers were between 40–60%, and Tm values were in the range of 59–61°C. Primer Express v2.0 (Thermo Fisher Scientific, MA, USA) software was used for designing primers with the above criteria. The specificity of amplification products of all InDel primer sequences was tested with the BLAST (http://genome-euro.ucsc.edu/cgi-bin/hgPcr). The web application (https://genetools.org/SNPCheck/) was used to check whether the designed primers corresponded to any SNP loci. AutoDimer v1 developed by the National Institute of Standards and Technology (NIST) was used for primer-dimer detection (https://strbase.nist.gov/AutoDimerHomepage/DownloadPage.htm). InDel primers were labeled by four different fluorescent dyes (JOE, 6-FAM, ATTO-550, and ATTO-565), respectively.

2.3 PCR Amplification and Genotyping

PCR amplification was performed using, 5 µL of 2X concentration Qiagen Multiplex PCR Master Mix (Qiagen, Germany), 3 µL of primer mix (primer concentrations ranges between 0.1–1.2 µM), 1 µL of distilled water, and 1 µL of total genomic DNA at 1 ng concentration. PCR amplification was performed on a GeneAmp 9700 (Thermo Fisher Scientific, MA, USA) under the following conditions: 15 min of initial denaturation at 95°C, followed by 30 cycles of 30 sec denaturation at 94°C, 90 sec annealing at 60°C, 45 sec extention at 72°C, and 60 min final extension at 72°C. For electrophoretic analysis of each amplified product, 10 µL of Hi-Di™ Formamide (Thermo Fisher Scientific, MA, USA) and 0.3 µl of GeneScan™ 500 LIZ® size standard (Thermo Fisher Scientific, MA, USA), and 1 µL of PCR product were mixed. The loading samples were denatured at 95°C for 3 minutes and kept on ice for 3 minutes. Then, the samples were run on ABI PRISM® 3130 (Thermo Fisher Scientific, MA, USA) genetic analyzer using POP-7 polymer with 36 cm capillaries. Samples were analyzed using GeneMapper v4.0 software (Thermo Fisher Scientific, MA, USA). Positive control DNA (K562, (Promega) was used in each run to test both amplification and multiplex genotyping performance.

2.4 Validation Studies of the 36-InDelplex Panel

In the validation studies of the 36-InDelplex; analysis threshold, dynamic range, sensitivity, stochastic threshold, repeatability, and reproducibility parameters were determined according to the SWGDAM guideline (SWGDAM, 2017) [20]. 10 negative control samples were run to determine the limit of detection (LOD) of the ABI PRISM® 3130 genetic analyzer device. Nine dilutions of positive control DNA K562 (15.625 pg, 31.25 pg, 0.0625 ng, 0.125 ng, 0.25 ng, 0.5 ng, 1 ng, 2 ng, 3 ng) were prepared to determine the dynamic range, sensitivity (LOQ-Limit of quantitation), and stochastic threshold parameters for the 36-InDelplex. Each input DNA was genotyped twice. The reproducibility parameters were evaluated by testing 10 samples with known profiles by different analysts at different times in the same laboratory and devices. The reproducibility parameters were determined by testing 10 samples with known profiles in a different laboratory and device by a different analyst.

DNA mixture samples were obtained by combining a female (K562) and a male (GML-Y) DNA sample at different concentrations (1:1, 1:5, 1:10, 1:20, 1:50 and 50:1 20:1, 10:1, 5:1). The major and minor profiles were analyzed on the 36-InDelplex.

DNA damage, which causes degradation, occurs under certain environmental conditions such as UV light, temperature, ionizing radiation, timing, and genotoxic chemicals. To measure the stability of the 36-InDelplex panel in degraded samples we tested DNA samples kept in high temperatures, humid environments, exposed to UV-C light and inhibitors.

For testing high temperatures, a DNA sample (10 µl of 10 ng/µl) was kept in the thermal block for 5 to 120 mins at 95˚C. For the humid environment; pieces of blood-stained cotton fabric formed using 10 ml of blood sample were placed suspended in a sterile falcon tube containing 10 ml of distilled water to provide humidity. Then, it was kept in the refrigerator at 4˚C for 1 to 16 weeks and 1 cm² piece of stained fabric was taken every week. For both high temperature and humid environments; pieces of blood-stained cotton fabric formed using 10 ml of blood sample were placed suspended in a sterile falcon tube containing 10 ml of distilled water to keep the humidity. Then, it was kept in an oven at 56˚C for 1 to 16 weeks and 1 cm² piece of stained fabric was taken every week. To determine the effects of UV exposure, 100 µl of 1 ng/µl DNA sample in a 1.5 ml microcentrifuge tube was exposed to UV (UV-C at 254 nm) for 5 minutes to 120 minutes.

To measure the performance of the 36-InDelplex panel in the presence of inhibitors in PCR; a series of concentration gradients of humic acid (50 ng/µl, 25 ng/µl, 12.5 ng/µl, 6.25 ng/µl), hematin (1000 µM, 500 µM, 250 µM, 125 µM) and indigo (4 mM, 3 mM, 2 mM, 1 mM) were examined.

2.5 Population Studies and Statistical Analysis

Allele frequencies, exact tests of Hardy-Weinberg equilibrium (HWE), pairwise fixation index (F_ST) values between Turkish and the reference populations were calculated using the Arlequin v.3.5.1.2 [21]. A series of forensic parameters including observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC), match probability (MP), power of exclusion (PE), power of discrimination (PD), and typical paternity index (TPI) were calculated with the PowerStats v1.2 software (Promega) Microsoft® Excel file. Mega X v.10.0.5 program was used for phylogenetic tree drawing.

3.1 Performance of the 36-InDelplex Panel

After optimization of the selected markers, primer design, and PCR conditions, the 36-InDelplex panel was successfully conctructed. The general information of the 36-InDelplex is indicated in Table 1. The allele lengths are between 2 to 10 bp and the maximum InDel length is located at rs16363 (ID34) locus. The PCR amplicons are ranged from 69 to 252 bp in length. Figure 1 shows the electrophoregram image of the K562 control DNA sample of the optimized 36-InDelplex panel. The control sample belongs to a female donor and the ID35 (rs2032678) locus is specific to the Y chromosome, thus it is not seen in this sample.

The analysis threshold (LOD) of the 36-InDelplex panel was determined as 112.39 RFU. The sensitivity of the 36-InDelplex panel was conducted by various concentrations of the positive control K562 DNAs (15.625 pg, 31.25 pg, 0.0625 ng, 0.125 ng, 0.25 ng, 0.5 ng, 1 ng, 2 ng, 3 ng). As shown in Fig. 2, full profiles were obtained until the input DNA dropped below 0.25 ng. Allele dropouts and peak imbalances were observed at peaks below 0.25 ng.

We examined heterozygote allele imbalance to calculate stochastic threshold values using nine dilutions of positive control DNA K562 (15.625 pg, 31.25 pg, 0.0625 ng, 0.125 ng, 0.25 ng, 0.5 ng, 1 ng, 2 ng, 3 ng). The stochastic threshold values were determined as 86.28 for 0.5 ng and 73.02 for 0.25 ng of DNAs. These results showed that when the ratio of sister allele peak heights drops below 0.25 ng, the peak balances may be disrupted and cannot be considered as a heterozygous allele peak. Ten samples were utilized in reproducibility and reproducibility studies and the same genotypes were observed for each sample. The results show that the novel 36-InDelplex panel is reproducible and reliable profiles can be obtained in different laboratories.

DNA mixtures at ratios of 1:1, 1:5, 1:10, 1:20, 1:50 and 50:1 20:1, 10:1, 5:1 made of a male (M) and a female (F) samples were analyzed using the 36plex panel. No allele dropout was observed at the ratios of 1:1, 1:5, and 5:1. However, allele dropouts were observed at 3 to 19 loci at the ratios of 1:10, 10:1, 1:20, and 20:1 (Table 2). As the DNA concentration difference between the major and minor contributors increased, the peaks of the minor contributor remained below the analysis threshold value and only the profile of the major contributor could be determined. Thus, the mixture sample could be reliably detected at a ratio of 5:1 with this panel. Jin et al. (2021) reported that in their 43 InDel panel mixture study, the full profiles were detected at 1:1, 2:1, 4:1, and 6:1 ratios, and the minor contributor could be detected at the maximum 6:1 mixture ratio [13].

3.2 Degradation Studies

Biological samples found at the crime scene are usually degraded because they are exposed to undesirable environments such as heat, light, moisture, or microorganisms. Due to the nuclease enzymes found in microorganisms, DNA is broken down into smaller components. Environmental factors (temperature, humidity, UV light) also cause the degradation of DNA by oxidative or hydrolytic effects [22]. It is important to evaluate the genotyping efficiency of the developed panel for degraded samples. Therefore, we evaluated the efficiency of the 36 InDelplex panel by testing artificial degraded samples which are exposed to high temperatures, humid environments, UV-C light, and inhibitors.

To evaluate the effect of temperature on the 36-InDelplex panel, a DNA sample was kept in a thermal block at 95˚C for time intervals of 5, 15, 30, 60, and 120 mins. The full genotype profiles are detected at the 5, 15, and 30 min intervals while RFU values of all loci are decreased by time (Fig. 3). We observed allele dropouts at 10 loci (rs2308101, rs144389514, rs4646006, rs28369942, rs2067147, rs3062629, rs2308072, rs10623496, rs1160965 and rs2067191) at the 60 mins and no alleles were detected at the 120 min (Supplementary Fig. 1). Machida et al. kept DNA isolates at 95˚C for 5, 15, 30, 60, 120, 240, and 360 mins intervals and then typed them with STR loci. They observed decreases in the RFU values of alleles starting from the 5th min. Additionally, it was reported that the RFU values of the alleles decreased largely at the end of 120 mins and there were allele dropouts in the following periods [23]. Li et al. evaluated the effect of the temperature at the 18 multi-InDels panels by keeping DNA isolates in a water bath at 100˚C for 0, 20, 40, 60, and 80 mins. Although all loci were observed in all time intervals, peak imbalances have begun to appear in the alleles after the 40th min [24]. The denaturation of DNA begins at 95˚C and the degradation becomes more permanent as time progresses. In both studies, as the exposure time at 95˚C increased, a directly proportional decrease in the RFU values was observed at the loci, and then allele and locus losses were experienced. Since InDel loci have shorter PCR products than STR loci, they are also resistant to degradation. Accordingly, this novel 36 InDelplex panel worked better than STR loci in heat-induced degradation.

To evaluate the effect of the humidity factor on the 36-InDelplex panel, samples were taken from the blood-stained fabric and kept in a humid environment at 4˚C in the refrigerator for periods of 1, 2, 4, 8, and 16 weeks. When the 1st, 2nd, 4th, 8th, and 16th weeks were examined, the RFU values of the alleles are decreased in general, while the rs1160965 and rs2067191 loci were dropped out starting from the 8th week (Fig. 3). At the 16th week, allele losses were observed in all loci (Supplementary Fig. 2). To evaluate the effects of both temperature and humidity factors on 36-InDelplex, the samples incubated in an oven at 56˚C on a blood-stained fabric were studied at 1, 2, 4, 8, and 16 weeks (Fig. 3). In general, RFU values were decreased in all weeks, while one locus (rs1160965) was dropped out after the 4th week. Two more loci (rs2307656 and rs2067191) were dropped out after 8 weeks. At the 16th week, no amplification was observed in any of the samples (Supplementary Fig. 3). These results showed that InDel loci are resistant to temperature and humidity.

We also assessed the effects of the UV factor on the 36-InDelplex panel. A DNA sample was exposed to UV-C (254 nm) for 5, 15, 30, 60, and 120 mins. Full profiles were obtained in all samples that were kept for 5, 15, 30, and 60 minutes, however, RFU values of all loci decreased by time. Allele dropout was observed at 12 loci (rs2032678, rs1610937, rs2307656, rs16363, rs2308101, rs28369942, rs16458, rs2308072, rs56168866, rs3062629, rs1160965, and rs2067191) at 120 minutes (Supplementary Fig. 4). In a similar study, SNP and STR loci were tested for UV exposed DNA samples which were kept at 254 nm for 5, 15, 30, 60, and 120 minutes [25]. According to the study, the RFU values are decreased at all alleles at the 5th minute in the STR loci allele dropouts were observed starting from the 15th minute, and that there were allele dropouts in all the alleles at the 120th minute. Although decreases in RFU values were observed at the 5th, 1 5th, 30th, and 60th minutes in the SNP loci, at the 120th minute, SNP typing could be performed in all loci except 3 SNP loci. This indicated that InDel and SNPs were less affected by UV degradation due to their shorter amplicon lengths than STRs.

We also assessed the tolerance for the PCR inhibitors on the 36-InDelplex panel. We mixed the control DNA with three different inhibitors (hematin, humic acid, and indigo) in various concentrations then samples were amplified and genotyped by this panel. For the hematin inhibitor 125, 250, 500, and 1000 µM of hematin was added to the PCR component and analyzed. Decreases in RFU values were observed in all loci at 125 and 250 µM concentrations. Allele dropout was observed at 6 loci (rs28369942, rs2067147, rs2308072, rs10623496, rs1160965, and rs2067191) at 500 µM concentrations, no amplification was observed at any loci at 1000 µM of hematin (Supplementary Fig. 5).

To evaluate the effect of the humic acid inhibitor on PCR, the humic acid was added to PCR components at different concentrations (6.25, 12.5, 25, and 50 ng/µl) and samples were amplified and analized with 36 InDelplex panel. At 6.25 and 12.5 ng/µl concentrations of humic acid, decreases in RFU values were observed at all loci, while at 25 and 50 ng/µl concentrations, 8 loci (rs2308112, rs28369942, rs2067147, rs2308072, rs16722, rs10623496, rs1160965, and rs2067191) were dropped out (Supplementary Fig. 6).

For the indigo inhibitor, genotyping was performed by adding indigo at 1, 2, 3, and 4 mM concentrations to PCR components. Although decreases in RFU values were observed in all loci at 1, 2, and 3 mM indigo concentrations, a full profile was obtained. Allele drop out was observed at only 1 locus (rs1160965) at 4 mM concentration (Supplementary Fig. 7). As a result, this study demonstrated that the 36-InDelplex panel showed good tolerance to different concentrations of the three inhibitors. Chen et al. conducted an inhibitor study with the 47 A-InDel (47 autosomal InDels and 2 Y-chromosome InDels) panel, they reported that full profiles can be obtained at < 100 µM hematin concentrations, < 50 ng/µl humic acid concentrations, and < 16 mM indigo concentration [18]. Our findings are similar to this study.

3.3 Population genetics

Allele frequencies and forensic parameters of the novel 36-InDelplex panel in the Turkish population were determined (Supplementary Table 1). After a Bonferonni correction (p = 0.00147), no significant deviations from Hardy-Weinberg equilibrium were found in all loci except for rs25549 (p < 0.00147). In our study, a triple allele was detected in the rs56168866 locus. The frequencies of the detected alleles were calculated as 0.124 del, 0.446 ins and 0.430 ins. The mean insertion allele frequency value for 34 autosomal InDel loci was calculated as 0.49, and the value ranged from a minimum of 0.31 (rs2308112) to a maximum of 0.642 (rs6480) (Supplementary Table 1). Allele frequency distributions of the 36-InDelplex panel were found to be in balance among alleles. Heterozygosity values (He) of 34 autosomal InDel loci were found the lowest at the rs1160981 locus (0.368) and the highest at the rs56168866 locus (0.601) with an average value of 0.485. The PD values ranged from 0.411 to 0.756, PE ranged from 0.29 to 0.112, and PM ranged from 0.589 to 0.244 (Supplementary Table 1). The combined PD, PE, and MP values of the 36-InDelplex panel were calculated as 0.999999999999988, 0.9987572351, 2.14x10^− 14, respectively. Thus, it has been determined that the discrimination power of the 36-InDelplex panel is over 99.99%, and the necessary exclusion and discrimination power can be achieved in forensic identification and determination of kinship. When we compare the CPD and CPE values of the Turkish population study of the DIPplex kit (Qiagen) by Duvenci et al., the 36-InDelplex panel has higher values and therefore is more advantageous in identification [26].

Pereira et al. (2009) developed an autosomal 38 InDel-multiplex panel [8]. Among the loci included in the panel, rs2308137, rs3397205, and rs1610919 markers are included in our 36-Indelplex panel. When the exclusion power values of the 3 loci common to the two studies are compared, the European population has similar discrimination power (CPD: 2.8x1014 for the European) to the Turkish population.

The 29-InDel panel has two of the common loci (rs1160981 and rs16671) with our panel. For the novel 36 InDelplex, PD of the rs1160981 locus was calculated as 0.532, for the 29-InDel panel it was the highest discriminating locus with a value of 0.650 at the Chinese population. While the PD of the rs16671 locus is 0.585 in the Chinese population, this value is 0.627 in the Turkish population. CPD and CPE values of the 29-InDel panel were calculated as 0.99999999990867/0.9930 and discriminant power of 99%, respectively, similar to our panel [27]. In another study, Tao et al. developed a multiplex kit containing 45-InDel (27 autosomal, 16-X chromosome, 2 Y-chromosome) loci for identification purposes [28]. In this study in the Chinese Han population, the cumulative discrimination power (CPD) for 27 autosomal InDel regions was calculated as 0.999999. The discriminative power of the common rs16458 locus with our study is higher in the Turkish population than in the Chinese Han population (Chinese Han, PD: 0.42 and Turkey PD: 0.632). Huang et al. developed a 32 InDel loci panel, in which the rs10590424 locus was the same as our study, and the discriminant power (PD) value was calculated as 0.62 for the Chinese population, and the PD was calculated as 0.596 for the Turkish population [19]. Pimenta et al. studied a multiplex of 40 InDel loci in a European population. The matching probability and exclusion power of the panel were calculated as 3.48 x10-17 and 0.9997, respectively [14].

Considering the number and characteristics of loci used in the panel, the discrimination power (CPD = 0.999999999999988) and exclusion power (CPE = 0.9987) values of our 36-Indelplex panel are at a level that can compete with other panels.

We estimated the genetic differentiation between Turkish and reference population groups from 1000 Genome Phase 3 data based on the autosomal 34 InDel loci of the panel. Table 3 shows the pairwise genetic distance (F_ST values) and corresponding p-values as calculated between the five reference populations (Africa, Europe, America, East Asia, and South Asia) and the Turkish population. The results of the pairwise genetic differences between tested population groups did not show significant differences. The results reveal that the novel 36 InDelplex panel constitute a globally suitable system for human identification purpose.

Table 3

Pairwise population F_ST estimates between the Turkish and reference populations. Below diagonal: F_ST values; above diagonal: corresponding p-values (the significance level was p < 0.001 after Bonferroni correction)
	Turkey	Africa	Europe	America	East Asia	South Asia
Turkey	-	0.00000+-0.0000	0.00000+-0.0000	0.00000+-0.0000	0.00000+-0.0000	0.00000+-0.0000
Africa	0.12395	-	0.00000+-0.0000	0.00000+-0.0000	0.00000+-0.0000	0.00000+-0.0000
Europe	0.02462	0.13864	-	0.00000+-0.0000	0.00000+-0.0000	0.00000+-0.0000
America	0.04458	0.13179	0.02709	-	0.00000+-0.0000	0.00000+-0.0000
East Asia	0.06439	0.15043	0.07179	0.07537	-	0.00000+-0.0000
South Asia	0.04370	0.13343	0.02240	0.02609	0.05364	-

The InDel polymorphism can be used in forensic identification as an alternative to STR and SNP, especially in degraded samples. The most important features sought for genetic markers used in forensic analyzes are both very stable structures and small amplicons. The low mutation rates and short amplicons of InDels have recently paved the way for their use in forensic genetics laboratories and are considered promising next-generation markers for forensic DNA analysis. In this study, a novel InDel identification panel (36-InDelplex) was established. This panel shows high polymorphism can be used in forensic identification and kinship analysis. After series of validation experiments, the 36-InDelplex panel was proven to be robust, highly sensitive and suitable for the analysis of challenging DNA samples.

Compliance with Ethical Standards:

Declaration of competing interest:

The authors declare have no financial or non-financial competing interests to disclose.

Ethical approval:

All procedures performed in studies involving human participants were in accordance with the ethical standards of the Clinical Research Ethics Committee of Istanbul University-Cerrahpasa, Cerrahpasa Medical Faculty (May 20, 2016, number 86669574-604.02.01-182593), and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. 250 healthy, Turkish citizens, who were not related to each other, were obtained informed consent.

Funding:

This study was supported by TÜBİTAK with project number 217S586 in the 1005 New Ideas and Products Research Support program. GF, OB, HK and FA have received this grand.

Acknowledgments:

Special thanks are due to the many hundreds of individuals who volunteered to give blood and saliva samples.

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Table 2 is available in the Supplementary Files section.

Table2.docx
Table 2. Detection of loci in the 36-InDelplex panel from various mixing ratios.
SupplementaryFiles.pdf

A Novel Panel of 36 Insertion/Deletion (InDel) Markers for Human Identification

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 DNA Samples

2.2 Locus Selection and Primer Design

2.3 PCR Amplification and Genotyping

2.4 Validation Studies of the 36-InDelplex Panel

2.5 Population Studies and Statistical Analysis

3. Results And Discussion

3.1 Performance of the 36-InDelplex Panel

3.2 Degradation Studies

3.3 Population genetics

4. Conclusions

Declarations

References

table

Supplementary Files

Status:

Version 1